Abstract: Dysregulation of protein kinase activity has been implicated in a number of diseases, including cancer, diabetes, and chronic inflammation. Therefore, protein kinases have emerged as one of the most important drug targets in modern drug discovery. These efforts have resulted in the development of a vast array of small molecule inhibitors that interact with the ATP-binding sites of protein kinases and are able to block their catalytic activities. Interestingly, many of these pharmacological agents exploit the conformational flexibility of the ATP-binding sites of protein kinases by binding to different active site conformations. ATP-competitive inhibitors that stabilize different ATP-binding site conformations of multi-domain protein kinases have been shown to divergently affect domains and interactions outside of their active sites. This proposal will explore how stabilizing different active site conformations of Src-Family Kinases affect their sub-cellular localization, inter-molecular interactions, and signaling behavior. A suite of chemical genetic and chemical proteomic tools is proposed to explore the consequences of conformation-selective, Src-Family Kinase inhibition in diverse cellular contexts.